The invention relates to absorption cells for microfluidic chemical analysis.
Microfluidic lab-on-a-chip (LOC) platforms1, 2) show considerable promise for the creation of robust miniaturised, high performance metrology systems with applications in diverse fields such as environmental analysis3, 4, potable and waste water, point of care diagnostics and many other physical, chemical and biological analyses. The technology allows the integration of many components and subsystems (e.g. fluidic control, mixers, lenses, light sources and detectors) in small footprint devices that could potentially be mass produced. Reduction in size enables reduction in power and reagent consumption making miniaturisation of a complete sensing system feasible. There are many applications to this technology, particularly in the development of remote in situ sensing systems for environmental analysis, and one area of importance is the measurement of ocean biogeochemistry.
Long term, coherent and synoptic observations of biogeochemical processes are of critical relevance for interpretation and prediction of the oceans' (and hence the earth's) response to elevated CO2 concentrations and climate change. Observations of oceanographic biogeochemical parameters are used to constrain biogeochemical models and understanding5-7 that in turn informs modelling of the ocean8 and earth system9. A promising approach for obtaining oceanographic biogeochemical data on enhanced spatial and temporal scales is to add biogeochemical sensors to existing networks of profiling floats or vehicles10. For long-term deployments these sensors should have high resolution and accuracy, negligible buoyancy change, low consumption of power and/or chemical reagents, and be physically small.
Colourimetric assays for determination of inorganic chemical concentrations (e.g. Nitrate/Nitrite11, Phosphate12, Iron13 and Manganese14 have long providence and are used widely in oceanography. Applied in laboratory15, shipboard16, and in situ analysis17-19 (i.e. in a submerged analytical system) they enable measurements over a wide measurement range including at low open ocean concentrations20.
The performance of colourimetric analytical systems is determined by both the fluidic and optical sub-systems. The optical system consists of an opto-fluidic cell in which the absorption of a fixed length of fluid is determined. The idealised relationship between the measured optical power, absorbance and chemical concentration is described by the Bouguer-Beer-Lambert law21-23. For mathematical simplicity we present here the exponential form of Beer's law23.Psample=P0e−αcl  Equation 1
Where Psample is the measured optical power, P0 the power of the optical source, α the absorption coefficient of the absorbing species, c the concentration of the absorbing species and l the effective length of the absorption cell. Care must also be taken in using the correct values of extinction coefficient from the literature as both natural and common logarithmic versions of the Bouguer-Beer-Lambert law are used.
The Bouguer-Beer-Lambert law only applies if a monochromatic light source is used, and if the concentration is low enough so that there is no interaction between molecules of the absorbing species. In this case, the absorbance due to the presence of the analyte and hence the concentration, is determined using Equation 2
                    A        =                              α            ⁢                                                  ⁢            cl                    =                      ln            ⁢                                          P                ref                                            P                sample                                                                        Equation        ⁢                                  ⁢        2            
Pref is frequently determined by measurement of a blank (i.e. a sample with no absorbing species)
The sensitivity of an idealised optical cell is maximum when the absorbance is equal to unity which implies an optimal cell length for a given absorption coefficient and concentration, which is determined as follows:
                                          ⅆ                          (                                                ⅆ                                      P                    sample                                                                    ⅆ                  C                                            )                                            ⅆ            l                          =                                            -                              α                2                                      ⁢                          lcP              0                        ⁢                          e                              (                                  -                  alc                                )                                              +                      α            ⁢                                                  ⁢                          P              0                        ⁢                          e                              (                                  -                  alc                                )                                                                        Equation        ⁢                                  ⁢        3            and at maximal sensitivity
            ⅆ              (                              ⅆ                          P              sample                                            ⅆ            C                          )                    ⅆ      l        =            0      ->      α        =                                        α            2                    ⁢          lc                ->                  α          ⁢                                          ⁢          lc                    =      1      
In practical implementations, monochromatic light is not used and P0 and α are wavelength dependent. In addition, ambient and stray light can arrive at the detector causing an offset; this light is unaffected by the concentration of the analyte. Therefore the measured power isP=∫P0(λ)e−α(λ)cldλ+Poffset  Equation 4
The spectral characteristics of the source and the extinction coefficient (even in the absence of stray light) imply that Equation 2 is not always applicable and if used incorrectly can result in a non-linear absorption measurement. For example Galli24 developed an analytical solution for a Gaussian source spectrum and a linear slope molecular extinction coefficient spectrum that demonstrates this departure from idealised behaviour.
Neglecting spectral effects, simple (and incorrect) application of Equation 2 without consideration of stray light causes a non-linear relationship between the effective absorption and concentration:
                    A        =                              ln            ⁢                                          P                ref                                            P                sample                                              =                      ln            ⁢                                          (                                                      P                    0                                    +                                      P                    offset                                                  )                                            (                                                                            P                      0                                        ⁢                                          e                                                                        -                          α                                                ⁢                                                                                                  ⁢                        cl                                                                              +                                      P                    offset                                                  )                                                                        Equation        ⁢                                  ⁢        5            
This deviation can be corrected if Poffset is known, and this can be determined directly by measuring the optical power when an opaque sample is placed in the absorption cell. A widely accepted method of obtaining high accuracy metrology is to eliminate stray light and to ensure the source, absorption and detector-sensitivity spectra convolve to give a wavelength independent response.
There have been many different approaches to integration and miniaturisation of microfluidic absorption cells. There are many examples of the use of thin and transparent materials to manufacture microchannel absorption cells25, but this approach is problematic. Whilst opto-fluidic integration with low dead volumes is possible the cell's absorption length is typically short, and stray light degrades performance. Kuswandi et al.26 and Hunt and Wilkinson25 recently reviewed opto-fluidic integration highlighting recent advances, including absorption cell design. Many systems use optical fibres for launching and collecting light from U-shaped (e.g.27) or Z shaped channels (e.g.28). Whilst the fibres' numerical aperture provides a degree of stray light rejection, alignment can be problematic. Complexity and optical power loss is also caused by coupling between fibres, sources and detectors. Grumann et al.29 used total internal reflection at an air interface in their polymeric devices to simplify coupling of out of plane sources and detectors to 10 mm long absorption cells. Stray light reduction relied on collimation of the laser source used. Lenses have been used to increase coupling efficiencies and to reduce stray light, but require complex fabrication for relatively short (500 μm) channels30. The use of liquid core waveguides (LCWs) enables both long path lengths and stray light rejection31-34. However LCWs can require complex fabrication and frequently rely on internal Teflon AF coatings that have poor long term performance (commercial macro Teflon AF based LCWs are supplied with a glass liner to prevent internal degradation). Multiple reflections can be used to increase effective absorption length to greater than the geometric length35, 36 though alignment and collimation remain problematic and only short effective path lengths are obtained. ARROW waveguide (e.g.37) and other structures facilitating absorption detection in the evanescent wave (e.g.38) but result in short interaction lengths for a given geometric length. Substrates doped with wavelength selective absorbent dyes that enable spectral filtering have been demonstrated in PDMS39, 40, 25 for optical filtering in fluorescence based systems. These arrangements have not been used for colourimetric assays and the control of stray light.
Despite these innovations simple, low-cost, robust absorption cells with long path lengths and low stray-light transmission remain elusive.